Allergy is a major health problem in countries where Western lifestyle is adapted. Furthermore, the prevalence of allergic disease is increasing in these countries. Although allergy in general may not be considered a life-threatening disease, asthma annually causes a significant number of deaths. An exceptional prevalence of about 30% in teenagers conveys a substantial loss in quality of life, working days and money, and warrants a classification among major health problems in the Western world.
Allergy is a complex disease. Many factors contribute to the sensitisation event. Among these is the susceptibility of the individual defined by an as yet insufficiently understood interplay between several genes. Another important factor is allergen exposure above certain thresholds. Several environmental factors may be important in the sensitisation process including pollution, childhood infections, parasite infections, intestinal microorganisms, etc. Once an individual is sensitised and the allergic immune response established, the presence of only minute amounts of allergen is efficiently translated into symptoms.
The natural course of allergic disease is usually accompanied by aggravation at two levels. Firstly, an progression of symptoms and disease severity, as well as disease progression, for example from hay fever to asthma. Secondly, dissemination in offending allergens most often occurs resulting in allergic multi-reactivity. Chronic inflammation leads to a general weakening of the mucosal defense mechanisms resulting in unspecific irritation and eventually destruction of the mucosal tissue. Infants may become sensitised primarily to foods, i.e. milk, resulting in eczema or gastrointestinal disorders; however, most often they outgrow these symptoms spontaneously. These infants are at risk of developing inhalation allergy later in their lives.
The most important allergen sources are found among the most prevalent particles of a certain size in the air we breathe. These sources are remarkably universal and include grass pollens and house dust mite faecal particles, which together are responsible for approximately 50% of all allergies. Of global importance are also animal danders, i.e. cat and dog dander, other pollens, such as mugwort pollens, and micro-fungi, such as Alternaria. On a regional basis yet other pollens may dominate, such as birch pollen in Northern and Central Europe, ragweed in the Eastern and Central United States, and Japanese cedar pollen in Japan. Insects, i.e. bee and wasp venoms, and foods each account for approximately 2% of all allergies.
All IgE-mediated allergic responses are directed towards proteins. In recent years the characterization of allergen molecules has advanced considerably facilitated by the application of modern techniques within molecular and structural biology. These studies have failed to demonstrate the existence of a common structural motif or a biological function shared by all allergens. It must be anticipated that any protein is a potential allergen, and that the most important allergens, i.e. the major allergens, are the most abundant proteins on airborne particles of a size facilitating their presentation to the immune system.
Allergy, i.e. type I hyper-sensitivity, is caused by an inappropriate immunological reaction to foreign non-pathogenic substances. Important clinical manifestations of allergy include asthma, hay fever, eczema, and gastro intestinal disorders. The allergic reaction is prompt and peaks within 20 minutes upon contact with the offending allergen often followed by a late reaction 8-24 hours later. Furthermore, the allergic reaction is specific in the sense that a particular individual is sensitised to particular allergen(s), whereas the individual does not necessarily show an allergic reaction to other substances known to cause allergic disease. The allergic phenotype is characterized by a pronounced inflammation of the mucosa of the target organ and by the presence of allergen specific antibody of the IgE class in the circulation and on the surfaced of mast-cells and basophils.
An allergic attack is initiated by the reaction of the foreign allergen with allergen specific IgE antibodies, when the antibodies are bound to high affinity IgE specific receptors on the surface of mast-cells and basophils. The mast-cells and basophils contain preformed mediators, i.e. histamine, tryptase, and other substances, which are released upon cross-linking of two or more receptor-bound IgE antibodies. IgE antibodies are cross-linked by the simultaneous binding of one allergen molecule. It therefore follows that a foreign substance having only one antibody binding epitope does not initiate an allergic reaction. The cross-linking of receptor bound IgE on the surface of mast-cells also leads to release of signaling molecules responsible for the attraction of eosinophils, allergen specific T-cells, and other types of cells to the site of the allergic response. These cells in interplay with allergen, IgE and effector cells, lead to a renewed flash of symptoms occurring 12-24 hours after allergen encounter.
Allergy disease management comprises diagnosis and treatment including-prophylactic treatments. Diagnosis of allergy is concerned with the demonstration of allergen specific IgE and identification of the allergen source. In many cases a careful anamnesis may be sufficient for the diagnosis of allergy and for the identification of the offending allergen source material. Most often, however, the diagnosis is supported by objective measures, such as skin prick test, blood test, or provocation test. The principle behind the blood test is the demonstration of specific IgE using an array of allergen sources. The therapeutic options fall in three major categories. The first opportunity is allergen avoidance or reduction of the exposure. Whereas allergen avoidance is obvious e.g. in the case of food allergens, it may be difficult or expensive, as for house dust mite allergens, or it may be impossible, as for pollen allergens. The second and most widely used therapeutic option is the prescription of symptomatic drugs. Symptomatic drugs are safe and efficient; however, they do not alter the natural cause of the disease, neither do they control the disease dissemination. The third therapeutic alternative is specific allergy vaccination i.e. specific immuno therapy that in most cases reduces or alleviates the allergic symptoms caused by the allergen in question.
Specific allergy vaccination is a causal treatment for allergic disease. It interferes with basic immunological mechanisms resulting in persistent improvement of the patients' immune status. Thus, the protective effect of specific allergy vaccination extends beyond the treatment period in contrast to symptomatic drug treatment. Some patients receiving the treatment are cured, and in addition, most patients experience a relief in disease severity, or at least an arrest in disease aggravation. Thus, specific allergy vaccination has preventive effects reducing the risk of hay fever developing into asthma, and reducing the risk of developing new sensitivities.
Specific allergy vaccination is, in spite of its virtues, not in widespread use, primarily for two reasons. One reason is the inconveniences associated with the traditional vaccination programme that comprises repeated vaccinations over a several months. The other reason is, more importantly, the risk of allergic side reactions. Ordinary vaccinations against infectious agents are efficiently performed using a single or a few high dose immunizations. This strategy, however, cannot be used for allergy vaccination since a pathological immune response is already ongoing.
Specific allergy vaccination is therefore carried out using multiple immunizations applied over an extended time period. The course is divided in two phases, the up dosing and the maintenance phase. In the up dosing phase increasing doses are applied, typically over a 16 week period, starting with minute doses. When the recommended maintenance dose is reached, this dose is applied for the maintenance phase, typically with injections every six weeks. Following each injection the patient must remain under medical attendance for 20-60 minutes due to the risk of anaphylactic side reactions, which in principle could be life-threatening. In addition, the clinic should be equipped to support emergency treatment. There is no doubt that eliminating the risk for allergic side reactions inherent in the vaccine would facilitate a more general use, possibly even enabling self vaccination at home.
The immunological mechanism underlying successful allergy vaccination is not known in detail. A specific immune response, such as the production of antibodies against a particular pathogen, is known as an adaptive immune response. This response can be distinguished from the innate immune response, which is an unspecific reaction towards pathogens.
An allergy vaccine is bound to address the adaptive immune response, which includes cells and molecules with antigen specificity, such as T-cells and the antibody producing B-cells. B-cells cannot mature into antibody producing cells without help from T-cells of the corresponding specificity. T-cells that participate in the stimulation of allergic immune responses are primarily of the Th2 type. Establishment of a new balance between Th1 and Th2 cells has been proposed to be beneficial and central to the immunological mechanism of specific allergy vaccination. Whether this is brought about by a reduction in Th2 cells, a shift from Th2 to Th1 cells, or an up-regulation of Th1 cells is controversial.
Recently, regulatory T-cells have been proposed to be important for the mechanism of allergy vaccination. According to this model regulatory T-cells, i.e. Th3 or Th1 cells, down-regulate both Th1 and Th2 cells of the corresponding antigen specificity. In spite of these ambiguities it is generally believed that an active vaccine must have the capacity to stimulate allergen specific T-cells, preferably TH1 cells.
With respect to allergen specific antibodies during the course of specific allergy vaccination an important observation is that in spite of clinical improvement there seem to be no corresponding change in IgE. Allergen specific IgE may provisionally rise early in treatment, but then falls back to pre-treatment levels during the treatment and may subsequently show a gradual decrease. Clinical improvement, on the other hand, is steadily increasing during the entire treatment period.
Another important observation is a large increase in allergen specific IgG early in the treatment period. Allergen specific IgG typically increases to concentrations hundreds or thousands times that of IgE. It has been proposed that allergen specific IgG is important for the immunological mechanism of specific allergy vaccination, since it can interfere or ‘block’ the interaction between IgE and the allergen. According to this model it is likely that IgG needs to undergo affinity maturation and/or increase in concentration by repeated immunizations before it can efficiently compete with IgE for binding to the allergen explaining the steady increase in clinical improvement observed during treatment.
The human immune system has two different ways of specific molecular recognition: via the T-cell receptor present on the surface of T-cells, and via the antibodies, which may be bound to the surface of the B-cells producing them, they may be free in solution, or they may be bound to specific receptors on the surface of a variety of cells in the immune system. Recognition via the T-cell receptor requires that the antigen is internalized and digested, and a fragment of the antigen is presented on the cell surface in complex with major histocompatibility complex (MHC), by an antigen presenting cell. The entire complex is recognized by the T-cell receptor.
The antigen presenting cell can be a professional antigen presenting cell, i.e. a macrophage, dendritic cell or a B-cell. Macrophages and dendritic cells ingest antigen by phagocytosis; whereas B-cells, capture antigen via surface bound antibody for subsequent internalisation and presentation. A T-cell epitope is a polypeptide fragment of 15-20 amino acids.
The B-cell epitope, i.e. antibody binding epitope, is different from the linear T-cell epitopes. Although antibodies that are raised in experimental animals by immunization in Freunds complete adjuvant may bind to linear polypeptide fragments, i.e. sequential epitopes, such antibodies are rarely raised during the natural response to allergens. Antibodies bind to a section of the surface of the correctly folded antigen molecule.
IgE epitopes of naturally occurring allergens are thus envisaged to consist of a number of surface exposed amino acids in a confined surface area that are supported by the backbone and the overall three-dimensional structure of the allergen. Such “epitope amino acids” are almost always found as either single amino acids or in clusters consisting of a few amino acids distributed over a large portion of the primary sequence of the allergen “brought together” on the surface upon folding of the molecule. The antibody binding epitope extends typically over approximately 800-900 Å2, but an even larger area is “masked” by the binding of the antibody. This means that upon binding of an antibody to an epitope, an area of up to 2000 Å2 becomes inaccessible to binding of other antibody molecules.
The affinity of the interaction between the antibody and the antigen is not only dependent on the attracting electrostatic forces, i.e. van der Waal's interactions, hydrogen bonding, and ion bridges, but also on the entropy gained by the almost complete expulsion of water molecules from the interface. Thus, the fit between the contours of the two molecular surfaces may be said to be an important parameter in defining the binding strength of the interaction. It follows that denaturation, i.e. unfolding, of the antigen (or allergen) usually destroys the antibody binding epitope, and binding of antibody will no longer be feasible in a physiological concentration range. It follows also, that if an amino acid in the epitope is substituted with another amino acid it will result in a reduction of the affinity of the interaction. The extent of the reduced antibody binding affinity depends on both the specific substituted amino acid and the antibody in question. It has e.g. been shown that one single amino acid substitution in an antigen can affect the interaction between antigen and antibody by a factor one thousand.
Attempts to improve vaccines for specific allergy vaccination by chemical modification have been performed for over 30 years and include multifarious approaches with one overall objective: to improve safety while not compromising efficacy; or in other words, to reduce IgE binding while not compromising immunogenicity.
Initial approaches included complete denaturation of the allergen. However, human trials have failed to show efficacy, which could be due to the non-efficient generation of a protective immune response. For the purpose of addressing T-cells in the allergic immune response, medium sized synthetic peptides (50-60 amino acids in length) were tested in human trials. Although the peptides showed some effect in very high doses, the trials were terminated due to the appearance of irregular side reactions. From these trials it seems that efficacy is compromised in the absence of antibody binding epitopes in the vaccine.
Other early attempts included chemical modification of allergen extracts either by acetylation (epitope masking), conjugation to large polymers (polyethyleneglycol), or polymerization into large so-called ‘allergoids’ by formaldehyde or glutaraldehyde. The former two concepts never entered human trials, whereas the ‘allergoids’ are currently in routine clinical use, however, without the benefit of improved safety. The reason for this is probably that chemical cross-linking randomly destroys some of the epitopes, which necessitates the use of higher doses for optimal efficacy, thereby increasing the level of allergic side effects approximately to the level observed using conventional allergy vaccines.
Approaches to improve allergy vaccines using genetic engineering include various strategies to disrupt the three-dimensional structure of the allergen vaccine molecules. Among these are substitutions of cysteine residues taking part in disulphide bridging. Another strategy is to assemble several naturally occurring amino acid substitutions into one molecule by site directed mutagenesis. A common characteristic of these strategies is to disrupt the tertiary structure of the vaccine, or at least not to consider preservation of the three-dimensional structure important.
Another commonly used approach has been to produce recombinant mutant variants of correctly folded naturally occurring allergens. The mutations are usually selected so as to reduce IgE affinity of the mutant protein.
WO 99/47608 discloses the introduction of articial amino acid substitutions into defined critical positions of the naturally occurring allergen while retaining the α-carbon backbone tertiary structure of the allergen.
WO 02/40676 discloses the introduction of at least 4 primary artificial amino acid substitutions into defined critical positions while retaining the α-carbon backbone tertiary structure of the allergen, wherein each primary mutation is spaced from another primary mutation by at least 15 Å and wherein at least one circular surface region with an area of 800 Å2 comprises no mutation.
J. Biomol. Struc. Dynamics (2000), vol. 17 pages 821-828 (Liang et al.) discloses transferring of a biological function of one protein to another by grafting the functional part of the protein to another appropriate scaffold protein. As a test system, the binding epitope of barstar, the inhibitor of barnase, is grafted onto a smaller molecule.
Protein Science (1994), vol. 3, p. 2351-2357 (Jin & Wells) discloses use of antigen-antibody complexes for studying protein-protein interactions. The binding of human growth hormone hGH to a monoclonal antibody (MAb 3) was studied. Five amino acids of hGH essential for the binding to MAb 3 was grafted onto a non-binding homologue of hGH, human placental lactogen (hPL), which has 86% sequence identity with hGH. Also, two additional framework mutations were introduced. The grafted hPL mutant bound to MAb 3 as well as hGH. Attempts to graft the epitope onto a scaffold protein having a homology with hGH of 23% were not successful.
J. Immunol (2001), vol. 166, p. 6057-6065 (King et al.) discloses hybrid insect venom allergens. The hybrids consist of an insertion of a small portion of the guest allergen of interest, Ves v 5, into a homologous host protein, Pol a 5. Ves v 5 and Pol a 5 have a sequence identity of 59% and a low degree of antigenic cross-reactivity. Hybrids are formed by inserting one portion of about 7 to about 50 amino acids into the allergen. The hybrids are useful for the mapping of B-cell epitope-containing regions of proteins and for use as immunotherapeutic reagents in man. This approach for generating allergen hybrids is however not appropriate for use in allergy vaccination strategies for the following reasons. Firstly, insertion of a peptide fragment from an insect allergen into an insect scaffold protein will in many cases lead to destabilization of the three-dimensional structure of the molecule. Unstable molecules are not suitable for use in vaccination. Secondly, as epitopes almost never consist of a single linear peptide fragment of the allergen, this approach is not suitable for “grafting” three-dimensional epitopes from allergens to scaffold proteins.